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Nucleic Acids Res
2012 Dec 01;4022:e173. doi: 10.1093/nar/gks765.
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Antisense morpholino targeting just upstream from a poly(A) tail junction of maternal mRNA removes the tail and inhibits translation.
Wada T
,
Hara M
,
Taneda T
,
Qingfu C
,
Takata R
,
Moro K
,
Takeda K
,
Kishimoto T
,
Handa H
.
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Gene downregulation by antisense morpholino oligonucleotides (MOs) is achieved by either hybridization around the translation initiation codon or by targeting the splice donor site. In the present study, an antisense MO method is introduced that uses a 25-mer MO against a region at least 40-nt upstream from a poly(A) tail junction in the 3''-untranslated region (UTR) of maternal mRNA. The MO removed the poly(A) tail and blocked zebrafish cdk9 (zcdk9) mRNA translation, showing functional mimicry between miRNA and MO. A PCR-based assay revealed MO-mediated specific poly(A) tail removal of zebrafish mRNAs, including those for cyclin B1, cyclin B2 and tbp. The MO activity targeting cyclins A and B mRNAs was validated in unfertilized starfish oocytes and eggs. The MO removed the elongated poly(A) tail from maternal matured mRNA. This antisense method introduces a new application for the targeted downregulation of maternal mRNAs in animal oocytes, eggs and early embryos.
Figure 1. Injection of cdk9, but not cdk9m, MOs into embryos inhibits polyadenylation and translation of zcdk9 mRNA during early development. (A) Target sequence within the zcdk9 mRNA 3′-UTR and MO hybridization positions. There are five mismatches in cdk9m MO. (B) Inhibition of zcdk9 poly(A) tail elongation in cdk9 MO-injected embryos. Embryos were collected at the times indicated. Total RNA was extracted from untreated (WT), cdk9 MO-injected and cdk9m MO-injected embryos. PAT assay was performed with PAT primers for cdk9, tbp and cyclin B1. PCR products were analyzed on a 2.2% agarose gel. Right side, length markers in bases. (C) Specific translation inhibition of zcdk9 mRNA by cdk9 MO, but not cdk9m MO. Embryos were collected at the indicated times. Western blotting was performed with extracts from untreated (lanes 1, 4, 7 and 10), cdk9 MO-injected (lanes 2, 5, 8 and 11) and cdk9m MO-injected embryos (lanes 3, 6, 9 and 12) by using the antibodies indicated. HeLa cell nuclear extracts (NEs) (lane 13) served as a control. Blots were probed with H-169, anti-zCdk9 and anti-actin. (D) The cdk9m MO (lanes 1 and 2), cdk9 MO (lanes 3 1nd 4) and 5′-cdk9 MO targeting a region around the first codon (lanes 5 and 6)-injected embryos were collected at 3 and 5 hpf. The extracts were subjected to western blotting by using antibodies indicated. (E and F) Reverse transcription using random (E) or oligo-dT primers (F), followed by PCR with primer sets for cdk9, biklf, tbp and actin, and analysis on a 2.2% agarose gel. Embryos were collected at the indicated times. Total RNA was extracted from untreated (WT: lanes 1–3), cdk9 MO-injected (cdk9 MO: lanes 4–6) and cdk9m MO-injected embryos (cdk9m MO: lanes 7–9).
Figure 2. The region 40-nt upstream from the zcdk9 3′-UTR end comprises the effective element for MO-mediated repression of zcdk9 mRNA. (A) Terminal sequence of the zcdk9 mRNA 3′-UTR and hybridization positions of antisense MOs. (B) Full repression of zcdk9 mRNA by injection of cdk9 MO, MO-5, MO-6 and MO-7. Embryos in which indicated MOs were injected (lanes 2 to 8) or uninjected (lane 1) were collected at 3 hpf. The total RNA was extracted, and a PAT assay was performed with PAT primers for cdk9 and tbp. (C) Extracts from embryos (5 hpf) in which indicated MOs were injected (lanes 2–8) or uninjected (lane 1) were subjected to 7.5% SDS–polyacrylamide gel electrophoresis. Blots were probed with anti-zCdk9, H-169 and anti-actin. HeLa cell NEs were also analyzed (lane 9) as a control.
Figure 3. Determination of the mRNA 3′-UTR end. (A) Schematic drawings indicate the method employed to determine the mRNA 3′-UTR end. (B) Experiments were performed with the total RNA used in Figure 2B. PCR products were visualized by ethidium bromide staining. (C) Results of DNA sequencing analysis of each cDNA derived from MO-injected embryos. Five clones were selected and the DNA sequences of their 3′-termini were aligned. A vertical line indicates a poly(A) tail junction of mRNA. The junction information was obtained from the NCBI nucleotide database (NM_212591.1.).
Figure 4. Specificity of polyadenylation inhibition by MO. (A) Sequences of cyclin B1 and B2 MOs. (B) Total RNA was extracted from 5 hpf embryos in which the indicated MOs were injected (lanes 2–4) or uninjected (lane 1). A PAT assay was performed with PAT primers for cdk9, cyclin B1 and cyclin B2. PCR using the actin primer set was performed with the cDNA produced in the PAT assay (actin). PCR products were analyzed on a 2.2% agarose gel. Right side, length markers in bases. (C) Total RNA was extracted from 5 hpf embryos in which the indicated MOs were injected (lanes 1–5) or uninjected (lane 6). A PAT assay was performed with PAT primers for cdk9, tbp and cyclin B1. The PCR products were analyzed on a 2.2% agarose gel. Right side, length markers in bases.
Figure 5. MO-mediated repression and recovery of starfish cyclin B mRNA. (A) Starfish oocyte maturation and experimental protocol. Immature oocytes (Im) were injected with MO, treated with 1-MeAde and subjected to western blot analysis. To isolate the RNA for the PAT assay in C, oocytes were recovered at 120 min after 1-MeAde treatment (pronuclear stage: PN). (B) Cyclin B mRNA 3′-UTR sequences adjacent to the poly(A) tail and sfcycB MO. (C) sfcycB MO inhibits polyadenylation of cyclin B, but not cyclin A, mRNAs. Total RNA was isolated from sfcycB MO-injected (lanes 3 and 7), control MO-injected (lanes 4 and 8) or uninjected PN oocytes (lanes 2 and 6), or from uninjected Im oocytes (lanes 1 and 5). Poly(A) tail lengths of cyclins A and B mRNAs were assessed by PAT. (D) Synthetic sfcyclin B mRNAs. sfcyclin B-zcyclin B1 3′-UTR: 5′-capped mRNA comprising the sfcyclin B coding region and zebrafish cyclin B1 3′-UTR (upper); sfcyclin B: 5′-capped mRNA comprising the cyclin B coding region without the 3′-UTR. (E) Chimeric mRNAs in D were translated by in vitro translation (IVT) (lanes 3 and 4). Synthesized cyclin B proteins assessed by western blot analysis with anti-cyclin B. Im lysate and IVT without mRNA were used as controls (lanes 1 and 2). (F) sfcyclin B-zcyclin B1 3′-UTR, but not sfcyclin B, restores cyclin B protein accumulation following MI exit in sfcycB MO-injected oocytes. Oocytes were injected with sfcycB MO and chimeric mRNA (left), and then were treated with 1-MeAde. Four oocytes were blotted with anti-cyclin B and anti-MAPK.
Figure 6. MO targeting to the 3′-UTR adjacent to the poly(A) tail induces deadenylation of the target mRNA. (A) Schematic representation of the experimental protocol. Im were treated with 1-MeAde. At 120 min later (PN120), oocytes in the pronuclear stage were injected with MO. After 90 min of incubation, the oocytes were recovered and total RNA was isolated (PN210). Total RNA was also isolated from oocytes of Im and PN120 as a control. (B) Sequences of the cyclin A 3′-UTR adjacent to the poly(A) tail and sfcycA MO. (C) sfcycA MO and sfcycB MO cause deadenylation of cyclins A and B mRNAs, respectively. According to A, total RNA was isolated from uninjected (−), or sfcycA MO-, sfcycB MO- or cdk9m MO-injected (control) oocytes. The poly(A) tail lengths of the cyclins A and B mRNAs of oocytes were monitored by the PAT assay. (D) Experimental schedule. (E) The shortened poly(A) tail does not support translation stimulation induced by U0126 addition. According to (D), eggs were injected with a mixture of sfcycA MO and sfcycB MO, treated with U0126 (at a final concentration of 10 µM) and then collected. Four oocytes were prepared for western blot analysis with anti-cyclin B and anti-MAPK antibodies.
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